Carbon Nanotube and Asbestos Exposures Induce Overlapping but Distinct Profiles of Lung Pathology in Non-Swiss Albino CF-1 Mice

Abstract

Carbon nanotubes (CNTs) are emerging as important occupational and environmental toxicants owing to their increasing prevalence and potential to be inhaled as airborne particles. CNTs are a concern because of their similarities to asbestos, which include fibrous morphology, high aspect ratio, and biopersistence. Limitations in research models have made it difficult to experimentally ascertain the risk of CNT exposures to humans and whether these may lead to lung diseases classically associated with asbestos, such as mesothelioma and fibrosis. In this study, we sought to comprehensively compare profiles of lung pathology in mice following repeated exposures to multiwall CNTs or crocidolite asbestos (CA). We show that both exposures resulted in granulomatous inflammation and increased interstitial collagen; CA exposures caused predominantly bronchoalveolar hyperplasia, whereas CNT exposures caused alveolar hyperplasia of type II pneumocytes (T2Ps). T2Ps isolated from CNT-exposed lungs were found to have upregulated proinflammatory genes, including interleukin 1ß (IL-1ß), in contrast to those from CA exposed. Immunostaining in tissue showed that while both toxicants increased IL-1ß protein expression in lung cells, T2P-specific IL-1ß increases were greater following CNT exposure. These results suggest related but distinct mechanisms of action by CNTs versus asbestos which may lead to different outcomes in the 2 exposure types.

Keywords: carbon nanotubes; comparative pathology; crocidolite asbestos; fiber toxicology; lung; mouse.

Fig. 1. Morphology and size distribution of carbon nanotubes (CNTs) and crocidolite asbestos (CA) materials used in this study

Left side: CNTs presented as loose tangled agglomerates with an average fiber diameter of 10.4 nm. Right side: CA was composed mostly of individual fibers with average diameter of 269.2 nm.

Fig 2. CNTs and crocidolite cause overlapping but distinct profiles of lung pathology

Vehicle-treated lungs exhibited little or no microscopic alterations. Both particle-treated groups showed granulomatous inflammation, composed chiefly of macrophages mixed with lymphocytes and low numbers of neutrophils. CNT-exposed lungs predominantly showed lymphoid tissue hyperplasia (interstitial and perivascular) and type II pneumocyte hyperplasia while crocidolite-treated lungs predominantly showed bronchoalveolar hyperplasia.

Fig 3. Extent and distribution of CA- or CNT-induced lung lesions

Hematoxylin and eosin (H&E) stains. Vehicle-treated lung, (A) 2× and (B) 4×, (C) CA-exposed lung showed granulomatous inflammation and epithelial hyperplasia predominantly at terminal airways and alveolar ducts, (C) 2× and (D) 4×. CNT-exposed lung showed diffuse alveolar hyperplasia in addition to granulomatous inflammation, (E) 2× and (F) 4×.

Fig 4. Regions of interest in CA- or CNT-exposed lung

H&E stains, 10×. (A) Vehicle treated lungs showed no change in airway or alveolar structure. (B) CA exposures caused hyperplasia of terminal bronchioles and alveolar ducts. (C) CNT exposures caused hyperplasia of alveolar epithelium and BALT/lymphoid tissue. CNT-laden macrophages are visible as darkly pigmented bodies.

Fig 5. CA- or CNT-induced lung epithelial hyperplasia

H&E stains, 20×. Vehicle-treated lungs showed little or no type II pneumocyte (T2P) and bronchoalveolar hyperplasia (A & B, respectively). CA-exposed lung showed modest T2P hyperplasia (C) but prominent bronchoalveolar hyperplasia (D). CNT-exposed lung showed more diffuse and severe T2P hyperplasia (E), while showing relatively little bronchoalveolar hyperplasia and peribronchial changes (F) in contrast with what is seen in CA-exposed lung.

Fig 6. CA- or CNT-induced interstitial remodeling

Masson’s trichrome stain, 40×. Vehicle-treated lungs showed no or minimal interstitial collagen staining (A). CA-exposed (B) and CNT-exposed (C) lungs showed increased interstitial collagen staining. Graph: Images of left lung sections stained with Masson’s trichrome were taken in bronchoalveolar regions (25 per slide). Collagen-specific color was isolated using color thresholding in ImageJ and resulting images were measured by mean pixel intensity. Distinct patterns of interstitial fibrosis following exposure were observed where (D) CA induced prominent perivascular and peribronchial fibrotic foci while (E) CNTs induced nodular fibrotic foci (arrows) in alveolar spaces following 8wk exposure to 300μg of either material.

Fig. 7. ProSPC⁺ type II pneumocytes (T2Ps) are increased in hyperplastic lesions induced by CNT exposure

Green: proSPC, Red: Ki-67, with DAPI. Pro-SPC⁺ cells were minimal in vehicle-treated lungs (A), increased in CA-treated (B), and especially prevalent in CNT-treated (C). Graph: proSPC⁺ cells were counted manually in lung sections and divided by number of alveoli in the field. CNT-exposed lungs had greatest numbers of type II cells/alveolus, with lesser increase seen with crocidolite exposure. Ki-67⁺ T2Ps were not observed. **p<.01.

Fig. 8. Prevalence of apoptotic T2Ps does not differ between CNT- and CA-induced hyperplastic lesions

Left column: proSPC-stained sections, right column: TUNEL-stained sections, 40× (Hematoxylin counterstain in both sets). Cytoplasm of T2Ps stained dark brown using DAB visualization of proSPC in (A) vehicle-treated, (C) CA-exposed, and (E) CNT-exposed alveolar epithelial lesions. TUNEL staining resulted in purple apoptotic nuclei in serial sections of the lesions (B, E, F, respectively). In general, significant prevalence or increases of apoptotic cells were not observed in any group. Serial sectioning allowed for TUNEL⁺ nuclei to be co-identified (where possible) as proSPC⁺ cells (see arrows, C and D, which denote a TUNEL⁺/proSPC⁻ cell). TUNEL⁺ T2P cells were uncommon and no trends were observed between exposure groups.

Fig. 9. CNT exposures more strongly induced pro-inflammatory mediators in type II pneumocytes (T2Ps) compared to crocidolite

RNA from isolated T2Ps was subjected to qRT-PCR. Values shown are the relative change from vehicle-treated samples.

Fig. 10. CNT and CA increase IL-1ß in lung cells, and CNT exposures induce more IL-1ß⁺ type II cells than CA

Green: proSPC, Red: IL-1ß, with DAPI. (A) Vehicle-treated lungs showed minimal IL-1ß staining. (B) CA-treated lungs showed IL-1ß increases which were not specific to T2Ps, whereas (C) CNT-treated lungs showed IL-1ß⁺ T2Ps. Upper graph: IL-1ß⁺ cells were counted manually and normalized to total number of nuclei. Changes between treatments are expressed as fold change over vehicle. Lower graph: Cells positive for both pro-SPC and IL-1ß were counted and normalized to total cells. Values are average number of double positive cells/field. *p<0.05, **P<0.01.